There are a variety of instruments and measurement techniques for diagnostic testing of materials related to human health, veterinary medical, environmental, biohazard, bioterrorism, agricultural commodity and food safety. Still, a solution for diagnostic testing and analysis of chemical or biological materials at the point of need remains limited. Diagnostic testing traditionally requires long response times to obtain meaningful data, involves expensive remote or cumbersome laboratory equipment that costs thousands of dollars located in a centralized laboratory, requires large sample sizes, utilizes multiple reagents, demands highly trained users, may require numerous steps, and/or involves significant direct and indirect costs. For instance, in both the veterinary and human diagnostic markets, most tests require that a specimen be collected from the patient and sent to the laboratory, but the results are not available for several hours or days later. As a result, the patient may leave the caregiver's office without confirmation of the diagnosis and the opportunity to begin immediate treatment.
Other problems related to portable devices include diagnostic results that are limited in sensitivity and reproducibility compared to in-laboratory testing. Fast response times are desirable and often critical to the identification of chemical and/or biological materials, such as in providing timely medical attention or in averting the spread or exposure of public health threats. Direct costs relate to the labor, procedures, and equipment required for each type of analysis. Indirect costs partially accrue from the delay time before actionable information can be obtained, e.g., in medical analyses or in the monitoring of chemical processes. Many experts believe that the simultaneous diagnosis and treatment enabled by an effective point of need diagnostic testing system would yield clinical, economic and social benefits.
Biosensors based on piezoelectric properties of materials have been used in detecting very small quantities of materials. Piezoelectric resonators used as sensors in such applications are sometimes called “micro-balances.” A piezoelectric resonator is typically constructed as a thin planar layer of crystalline piezoelectric material sandwiched between two electrode layers. When used as a sensor, the resonator is coated with a binding layer which, when exposed to the material being detected, allows the material to bind to the surface of the resonator. Modern resonators are fabricated using MEMS techniques and can be constructed to be so small that their resonant frequency is on the gigahertz scale. In general, resonators having higher resonant frequencies are more sensitive.
The conventional way of detecting the amount of the material bound on the surface of a sensing resonator is to operate the resonator as an oscillator at its resonant frequency. As the material being detected binds on the resonator surface, the mass of the resonator increases and the resonant frequency of oscillation is consequently reduced. The change in the resonant frequency of the resonator over time, presumably caused by the binding of the material on the resonator surface, is indicative of the amount of the material that is bound on the resonator or the rate at which the material accumulates on the resonator surface. From this data, a concentration of the material of interest, or analyte, present in the sample can be computed.
Conventionally, biosensors of this type generally include an assembly in which an intrinsic biosensor is surrounded by at least one fluidic channel, which is coupled to a sample reservoir for presenting a sample to the biosensor in a controlled manner. Most conventional biosensor configurations also include a mechanism for controlling and maintaining a desired temperature of the sample as it is presented to the intrinsic biosensor. In operation, the sample is drawn through the fluidic channel and across the intrinsic biosensor by application of vacuum or similar actuation pressure at a vacuum port. Oftentimes, the sample must be refined prior to introduction into the assembly by the addition of buffer or removal of certain elements such as whole cells or other particulates, which can interfere with the accuracy of the measurements. This refining step can be cumbersome and costly, making such measurements impractical for field applications. In addition, the introduction of the sample changes the physical environment in which the measurement is made. For example, the resonator oscillates at different resonant frequencies when exposed to a liquid sample versus prior to exposure when the resonator is in free air oscillation due to differences in viscosity between the two fluids.
Another approach involves stabilizing the measurement environment prior to introduction of the sample reagent. For instance, one type of instrument has two separate fluid reservoirs in which the first reservoir contains a buffer solution and the second reservoir contains the sample reagent. In operation, the buffer solution is introduced to the biosensor first, and the system is allowed to stabilize. Next, a valve switches to the sample reagent and measurements are made. The introduction of the buffer solution permits the sensor's oscillation to be tuned, or adjusted in accordance with the viscosity and temperature conditions of the buffer, which closely approximates the viscosity and temperature of the sample. The objective of this tuning is to operate the resonator as close as possible to its ideal resonant frequency for maximum sensitivity. One drawback of this approach is the introduced complexities and processing time requirements, which can result in expensive, error-prone, and time-consuming test results.
Another challenge faced by designers of resonant biosensors is in making quantifiable concentration measurements on highly concentrated samples. One trade-off of having the increased sensitivity of micro-scale (or smaller) resonators is their susceptibility to becoming rapidly saturated with analyte. In certain tests, a sensor can become saturated in a matter of seconds or even in a fraction of a second. In this case, although the sensor can operate as a simple detector of the presence of analyte, it cannot accurately measure the rate at which the analyte binds to the sensor, commonly referred to as binding kinetics. Measurement of binding kinetics is needed for quantification of the concentration of the analyte, can be impossible to obtain with any suitable accuracy or repeatability in highly concentrated samples using conventional techniques. Moreover, when analyte begins to bind to the resonator in significant quantity immediately from the moment that the resonant sensor is introduced to the sample, the instrument may not have the time to achieve its required stabilization prior to the taking of measurements, or to perform tuning of the resonator either before or after the taking of measurements, thereby further exacerbating the problem.
In view of the above, a practical solution is needed to enable the measurement of binding kinetics in sensitive instruments, particularly early binding kinetics from the time the sample is introduced to the sensor. Additionally, it would be desirable to perform such measurements without having to undertake the complexity of stabilizing the resonating sensor using a buffer solution or specially-refined sample.